Peripheral tolerance is now known to involve multiple mechanisms that control deletion and hypo-responsiveness of effector T cells, and can be strongly controlled by natural and adaptive regulatory T cells (Treg). We have been studying one member of the TNFR family, OX40 (CD134), whose expression is inducible on nave T cells and we have gained much data over the past few years that relate to the costimulatory ability of this receptor. Specifically, OX40 can signal through several pathways including PI-3-kinase, Akt, PKC8, and NF-?B, and target a number of intracellular molecules such as Bcl-2 and Bcl-xL that control apoptosis, or survivin and aurora B kinase that control cell cycle progression. As such, under non- tolerizing conditions, OX40 can function to prolong clonal expansion of effector CD4 and CD8 cells, enhance differentiation and cytokine secretion, and promote T cell memory. Studies in vivo have reinforced the notion that OX40 is strongly involved in the balance between tolerance and immunity. OX40 is also highly expressed on CD25+ natural Treg, and we present data in several in vitro and in vivo systems that suggest that OX40 can also impact positively and negatively the development or persistence of inducible antigen-specific (adaptive) Treg in response to TGF-?. In this competitive renewal, we will pursue how OX40 controls the balance in activity between non-regulatory and regulatory CD4 cells, and determine whether OX40 can play a dual role in both promoting or antagonizing Foxp3+ Treg. We will investigate how OX40 controls signaling through the TGF-?R in nave CD25- CD4 cells and whether OX40 suppresses Smad2/3 activation that leads to Foxp3 transcription, and whether OX40 promotes Smad7 expression that might suppress Foxp3 and antagonize Treg development. Further studies will elucidate the signaling pathways from OX40 that suppress adaptive Treg generation and/or activity focusing on Akt and the canonical and non-canonical pathways of NF-?B. We will also investigate whether OX40 can function to promote growth of adaptive and natural Treg by targeting Bcl-2 anti-apoptotic molecules or cell cycle molecules such as survivin and aurora B. Studies will be extended in vivo using retroviral and lentiviral transduction systems to determine how OX40 and its signaling targets control Treg generation and activity, and a tolerance model involving lung inflammation will be utilized to prove the importance of OX40 and its signaling pathways in controlling regulatory T cells and to understand the synergistic inflammatory signals that are required for OX40 to function to modulate Treg. PUBLIC HEALTH RELEVANCE Costimulatory molecules and their receptors are central to any immune response, providing signals that amplify the responses of T cells. OX40 signaling to T cells can prevent and even reverse a process termed tolerance, showing that OX40 is a potential target for therapeutic interventions in immune-mediated disorders where immunity is weak. OX40 is also then a target for intervention in a situation where tolerance is beneficial such as in autoimmune diseases. By understanding how OX40 controls the balance of effector/pathogenic T cells and regulatory T cells that suppress immune responses, this will aid in future strategies that might target human disease.